Schmallenberg virus : Pathogenesis of the infection in domestic ruminants and circulation in wild ruminants

HAL Id: tel-01310151

https://tel.archives-ouvertes.fr/tel-01310151

Submitted on 2 May 2016

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Schmallenberg virus : Pathogenesis of the infection in domestic ruminants and circulation in wild ruminants

Eve Laloy

To cite this version:

Eve Laloy. Schmallenberg virus : Pathogenesis of the infection in domestic ruminants and circulation

in wild ruminants. Agricultural sciences. Université Paris Sud - Paris XI, 2015. English. �NNT :

2015PA114836�. �tel-01310151�

UNIVERSITÉ PARIS-SUD 11

ECOLE DOCTORALE :

INNOVATION THÉRAPEUTIQUE : DU FONDAMENTAL A L’APPLIQUÉ PÔLE : MICROBIOLOGIE / THERAPEUTIQUES ANTIINFECTIEUSES

DISCIPLINE : Virologie

ANNÉE 2015 - 2016 SÉRIE DOCTORAT N°

THÈSE DE DOCTORAT

soutenue le 29/09/2015 par

Eve LALOY

Virus Schmallenberg : Pathogenèse de l’infection chez les ruminants domestiques et circulation chez les ruminants sauvages

Schmallenberg virus: Pathogenesis of the infection in domestic ruminants and circulation in wild ruminants

Directeur de thèse :

Composition du jury :

Président du jury : Prénom NOM

Rapporteurs : Martin BEER Professeur, Directeur de l’Institute of Diagnostic Virology au Friedrich-Loeffler-Institut

Sandra SCHOLES Consultante en Anatomie Pathologique Vétérinaire, Edinburgh Disease Surveillance Centre

Examinateurs : Stéphane BERTAGNOLI Professeur, Ecole Nationale Vétérinaire de Toulouse Audrey ESCLATINE Professeur, Faculté de Pharmacie de Châtenay-Malabry Laurence FIETTE Chercheur, Institut Pasteur

Nadia HADDAD Professeur, Ecole Nationale Vétérinaire d’Alfort Yves MILLEMANN Professeur, Ecole Nationale Vétérinaire d’Alfort Membres invités : Emmanuel BREARD Chercheur, UMR 1161 ANSES/INRA/ENVA Virologie

Nathalie CORDONNIER Maître de Conférences, Ecole Nationale Vétérinaire d’Alfort

i Ce qu’un mot ne sait pas, un autre le révèle ; Les mots heurtent le front comme l’eau le récif ; Ils fourmillent, ouvrant dans notre esprit pensif Des griffes ou des mains, et quelques-uns des ailes

Victor Hugo

ii

iii Acknowledgments

First, I would like to thank the members of the defense committee for this PhD thesis:

Sandra Scholes and Martin Beer, who kindly accepted to review my manuscript. I feel very sorry I am not Shakespeare.

Nadia Haddad and Yves Millemann for their enthusiasm and their long-lasting interest.

Audrey Esclatine and Stéphane Bertagnoli, I am in debt to you for coming to my defense.

Laurence Fiette, you have always been an inspiration to me.

Nathalie Cordonnier and Emmanuel Bréard, my co-supervisors, who strongly supported me all along the last three years.

Stéphan Zientara, my main supervisor, without your endless energy and dedication this thesis would not exist.

I also wish to thank the members of the doctoral supervisory committee. Claire Ponsart, Nathalie Cordonnier, Nadia Haddad, Yves Millemann, Emmanuel Bréard, Stéphan Zientara, you have spent time to check my PhD was going the right way.

I am grateful to the members of the five-country consortium of veterinary research institutes that conducted studies on Schmallenberg virus, especially the coordinator Wim Van der Poel, and Norbert Stockhofe (Central Veterinary Institute, the Netherlands), for productive discussions.

Merci à mes collègues Nathalie Cordonnier, Edouard Reyes-Gomez et Jean-Jacques Fontaine pour votre soutien ces dernières années. Sans vos encouragements et vos heures supplémentaires, il

m’aurait été impossible de mener à bien ce travail.

Merci à Gaston, mon « père de thèse » à défaut d’être mon père de clinique. Ta disponibilité et ta bienveillance sont précieuses.

Je souhaite remercier toutes les autres personnes qui ont collaboré à ce travail :

Les membres de l’UMR 1161 Virologie ANSES-INRA-ENVA, qui m’ont accueillie à bras

ouverts et m’ont enveloppée de cette bonne ambiance, unique. En particulier l’équipe EVIR : Stéphan,

Damien, Julie, Corinne, Manu, Cyril, Cindy, Annabelle, Greg, Aurore, Gaston, Virginie, Alexandra :

travailler avec vous est un plaisir et cela devait être écrit. Merci à mes co-doctorantes, Anne-Laure,

Chloé et Julie, pour votre patience face à mes innombrables interrogations. Merci à Typhaine,

ancienne étudiante à l’ENVA, que j’ai eu plaisir à encadrer en thèse de doctorat vétérinaire.

iv Benoît Durand (Unité Epidémiologie de l’ANSES) et Claire Ponsart (Unité Zoonoses

bactériennes de l’ANSES), qui ont piloté R avec un entrain hors du commun.

Isabelle Lagrange (Laboratoire d’hématologie ENVA) qui m’a accueillie dans son laboratoire avec mes tubes et mes questions.

Vincent Mauffré et Fabienne Constant (UMR Biologie de la reproduction INRA-ENVA) pour leurs précieuses suggestions.

Sophie Rossi de l’ONCFS pour sa patience et ses enseignements.

L’équipe du laboratoire d’Anatomie pathologique de l’ENVA : Agnès, Sophie, Patricia, Hélène et Hélène, Fara, Narcisse, Jean-Luc, Sandrine et les résidents. Merci pour votre soutien toutes ces années. J’adresse toute ma gratitude à Sophie pour les discussions scientifiques régulières que nous avons entretenues. Mille mercis à Hélène H. qui m’a accueillie sous son toit pendant mes passages à l’INRA de Nouzilly. Merci à Haintso, qui a accepté de travailler sur les tissus de chèvres lors de son stage au laboratoire.

Parmi les membres de l’INRA de Nouzilly :

Sascha Trapp, thank you for your dedication to the SBV project and your words of advice. A toute l’équipe de la PFIE, merci pour votre accueil chaleureux et votre investissement. Céline, Mickaël, Rémi, Sylvain, Didier, j’ai apprécié vos encouragements et salue votre disponibilité. Merci à Marie, en stage à la PFIE, pour sa participation.

Les membres du Laboratoire National de Contrôle des Reproducteurs, en particulier Maxime Raimbourg et Nathalie Pozzi : merci pour votre implication dont la constance mérite d’être soulignée.

L’équipe de la Ménagerie du Jardin des Plantes, en particulier Norin Chai qui s’est démené pour financer notre projet sur les animaux de zoo et le valoriser. Merci à Claire Réjaud pour sa réactivité au long du projet. Merci à Cindy, étudiante de l’ENVT, pour son engagement sur ce projet malgré la distance géographique qui nous a souvent séparées.

I wish to thank Jacques Kaandorp from SafariPark Beekse Bergen for its contribution to this work.

Je remercie enfin mes amis, mes proches et mon très proche, qui m’ont aidé à préserver cette petite

flamme intérieure, malgré les coups de vent.

v Virus Schmallenberg : Pathogenèse de l’infection chez les ruminants domestiques et circulation chez les ruminants sauvages

Le virus Schmallenberg (SBV) appartient au genre Orthobunyavirus, au sein de la famille des Bunyaviridae. Ce nouveau virus, découvert en 2011 au nord-ouest de l’Europe, affecte les ruminants domestiques. Il est responsable de signes cliniques discrets chez les adultes et de malformations congénitales chez les nouveau-nés. Ces travaux de thèse s’inscrivent dans les projets d’étude de la pathogenèse de l’infection à SBV et de l’épidémiologie de la maladie, dans le cadre d’un programme de recherche européen sur le virus. Ce manuscrit inclut de nouvelles données, telles les cinétiques de la virémie et de la séroconversion chez les ovins et caprins, après infection expérimentale par SBV. La possibilité d’infection par SBV par voie vaginale est démontrée expérimentalement chez la chèvre. Après infection expérimentale de chèvres gestantes entre 28 et 42 jours de gestation, une mortalité fœtale ou des lésions du système nerveux central des fœtus peuvent survenir. Enfin, la sensibilité de plusieurs espèces de ruminants sauvages et exotiques de parcs zoologiques vis-à-vis de SBV est démontrée pour la première fois.

Mots clés : Virologie ; Arbovirus ; Orthobunyavirus ; Virus Schmallenberg ; Infection

expérimentale ; Reproduction ; Caprins ; Ovins ; Faune sauvage ; Ruminants exotiques ; Parc

zoologique.

vi Schmallenberg virus: Pathogenesis of the infection in domestic ruminants and

circulation in wild ruminants

Schmallenberg virus (SBV) belongs to the genus Orthobunyavirus in the family Bunyaviridae. This new virus was discovered in 2011 in Northwestern Europe in domestic ruminants. Infection by SBV is associated with mild clinical signs in adult and congenital malformations in the progeny. In the scope of the European research program on SBV in the pathogenesis and epidemiology areas, the works included in this thesis provide new data about SBV infection in livestock and wild and exotic ruminants. The kinetics of viremia and seroconversion after experimental SBV infection are described in sheep and goats. This manuscript includes evidence of SBV infection via vaginal route in goats. Experimental SBV infection in pregnant goats between 28 and 42 days of gestation can lead to death or central nervous system lesions in fetuses. Evidence of susceptibility to SBV in several species of wild and exotic ruminants kept in zoos is described for the first time.

Keywords : Virology; Arbovirus; Orthobunyavirus; Schmallenberg virus; Experimental

infection; Reproduction; Caprine; Ovine; Wildlife; Exotic ruminants; Zoological park.

vii Laboratoire de rattachement :

UMR Virologie 1161 ANSES-INRA-ENVA ANSES

23 avenue du Général de Gaulle 94704 Maisons-Alfort

France

viii

ix Table of contents

1 Introduction ... 1

2 State of the Art ... 4

2.1 Discovery of a new virus ... 4

2.1.1 History ... 4

2.1.2 Structure... 4

2.1.3 Phylogeny ... 6

2.2 Epidemiology ... 9

2.2.1 Susceptible species ... 9

2.2.2 Transmission ... 12

2.2.3 Geographical repartition ... 15

2.2.4 Risk factors ... 16

2.3 Clinical signs and lesions in affected animals ... 18

2.3.1 Non-pregnant adults ... 18

2.3.2 Pregnant females and their offspring... 19

2.3.3 Hypotheses on the pathogenesis of the lesions in fetuses and newborns ... 24

2.4 Impact on livestock farming, on wild ruminants ... 30

2.5 Diagnostics and preventive measures ... 32

2.5.1 Diagnostics ... 32

2.5.2 Preventive measures ... 37

3 Aims of the Thesis ... 41

4 Experiments ... 43

4.1 Pathogenesis in domestic ruminants ... 43

4.1.1 Infection of adult sheep (published paper) ... 43

4.1.2 Infection of adult, non-pregnant goats ... 47

4.1.3 Infection of adult goats around the time of insemination and during pregnancy ... 51

4.2 Circulation in wild and exotic ruminants ... 66

4.2.1 Free-ranging wild ruminants in France ... 66

4.2.2 Wild and exotic ruminants kept in zoos in France and the Netherlands ... 70

x

5 Discussion ... 74

5.1 Pathogenesis of the infection with SBV: hypotheses drawn from experimental infection in domestic ruminants ... 74

5.1.1 Pathogenesis of the infection in males and non-pregnant females ... 74

5.1.2 Pathogenesis of the infection in pregnant females ... 79

5.2 Rapid and broad dissemination of the virus among wild and exotic ruminants ... 89

5.2.1 A quick spread in many species and in various ecosystems ... 89

5.2.2 Wild ruminants: do they play a role in SBV dissemination in domestic ruminants? ... 91

5.3 Consequences in the field: impact and preventive measures ... 92

5.3.1 Domestic ruminants ... 92

5.3.2 Wild and exotic ruminants ... 93

6 Future directions ... 95

6.1 Pathogenesis of the infection in goats ... 95

6.2 SBV circulation in wild and exotic ruminants ... 96

7 Conclusion ... 98

8 References ... 100

9 Appendix ...

xi List of figures

Figure 1. Morphology of SBV: schematic drawing on the left, electronic microscopy on the right (bar = 100

nm). ... 5

Figure 2. Phylogenetic relationships of Simbu serogroup viruses for the M (A), L (B), and S (C) coding regions.7 Figure 3. Regions (NUTS2) with at least one SBV herd confirmed by direct detection by period of first report (Afonso et al., 2014). ... 15

Figure 4. Gross and histological lesions from SBV-infected fetuses (Herder et al., 2012). ... 22

Figure 5. Hypothetic consequences of SBV infection in pregnant goats and ewes depending on the stage of gestation. ... 26

Figure 6. Experimental design. ... 51

Figure 7. Evolution of the LH concentration in goats from groups A and D after sponge removal. ... 57

Figure 8. Evolution of the progesterone concentration (P4, ng/mL) in serum in groups A, B and D. ... 58

Figure 9. Evolution of the progesterone concentration (P4, ng/mL) in serum in groups C and D’. ... 59

Figure 10. Hypothetical pathogenesis after SBV infection in adult non-pregnant ruminants. ... 78

Figure 11. Outcomes and pathogenesis after SBV infection in adult pregnant goat. ... 88

xii List of tables

Table 1. Detection of SBV infection in wild and exotic ruminants from the wild or kept in captivity. ... 10

Table 2. Lesions in fetuses and newborns associated with spontaneous SBV infection in domestic ruminants. ... 21

Table 3. Lesions in fetuses and newborns associated with AKAV infection in domestic ruminants. ... 23

Table 4. Impact of SBV infection in domestic ruminants in Europe and France, by species. ... 31

Table 5. Pregnancy outcomes per group. ... 55

Table 6. In a subset of organs containing SBV RNA without associated lesions in experimentally infected goat

fetuses: lesions found in SBV-infected neonates in the field, arguments for and against a causal link

between SBV RNA presence and lesion formation. ... 86

xiii Abbreviations

ANSES: Agence Nationale de Sécurité sanitaire de l’alimentation, de l’environnement et du travail

AKAV: Akabane virus BDV: Border disease virus

BHK cells: baby hamster kidney cells (cell culture) BTV: Bluetongue virus

BVDV: Bovine viral diarrhea virus CNS: central nervous system

CPT-Tert cells: sheep choroid plexus cells (cell culture) CVV: Cache Valley virus

dg: day(s) of gestation dpi: day(s) post-infection

ENVA: Ecole Nationale Vétérinaire d’Alfort HE: Hematoxylin-eosin

HES: Hematoxylin-eosin-saffron

INRA: Institut Nationale de la Recherche Agronomique IHC: immunohistochemistry

ISH: in situ hybridization

KC cells: Culicoides variipennis larvae cells (cell culture)

OIE: World Organization for Animal Health (historical acronym from the French “Office International des Epizooties”30)

SBV: Schmallenberg virus

TCID

: 50% tissue culture infectious doses

xiv

1

1 I NTRODUCTION

Schmallenberg virus (SBV), belonging to the genus Orthobunyavirus in the family Bunyaviridae, was discovered in 2011 in Northwestern Europe (Hoffmann et al., 2012b), five years after the emergence of Bluetongue Virus serotype 8 (BTV-8) in the same area (Wilson and Mellor, 2009). Similarly to BTV, SBV is an arbovirus transmitted by midges of the genus Culicoides (De Regge et al., 2012) and cause clinical disease in domestic ruminants (van den Brom et al., 2012; Hoffmann et al., 2012b).

The disease associated with SBV is an example of an emerging disease, as defined by the World Organization for Animal Health (OIE): ‘a new occurrence in an animal of a disease, infection or infestation, causing a significant impact on animal or public health resulting from:

(i) a change of a known pathogenic agent or its spread to a new geographic area or species; or (ii) a previously unrecognized pathogenic agent or disease diagnosed for the first time’ (OIE, 2015). As a reaction to this emerging disease, the European Commission identified in February 2012 the major areas for which research was needed prior to decision making about regulation and control. These areas were the pathogenesis, the epidemiology and the diagnostic of SBV-associated disease. A consortium gathering laboratories from Belgium, Germany, France, the Netherlands and the United Kingdom has performed a large part of the corresponding studies.

The studies presented in this PhD thesis have been carried out in the scope of the consortium research program in the pathogenesis and epidemiology areas. I was involved in pathogenesis studies focusing on small domestic ruminants, especially the goat - as determined by the task allocation planned by the Consortium. The epidemiologic studies included in this thesis deal with exposure of wild and exotic ruminants to SBV.

In chapter 2, a review about SBV will provide general knowledge about the virus, a state of

the art about epidemiology of the disease as well as work hypotheses about pathogenesis of

the lesions associated with SBV. The objectives of the thesis will be detailed in chapter 3,

followed by the corresponding works in chapter 4. A general discussion and future directions

will be provided in chapter 5 and chapter 6, respectively.

2

3

4

2 S TATE OF THE A RT

2.1 Discovery of a new virus

2.1.1 History

In summer and fall 2011, an unidentified disease was reported in dairy cattle in Germany and in the Netherlands. The clinical signs were not specific, including fever, decreased milk production and diarrhea. No known agent could be identified in the affected cattle. Sequences of a new virus were then detected after metagenomic analysis on blood samples. This virus was named “Schmallenberg virus” after the city of Schmallenberg from where the first positive samples came. Phylogenetic analyses showed that the virus belonged to the Simbu serogroup in the genus Orthobunyavirus. Viruses of this serogroup had not been identified previously in Europe (Hoffmann et al., 2012b).

A retrospective study on ruminant brain tissues archived from 1961 to 2010 in Germany showed no SBV RNA or antigen in these tissues by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively (Gerhauser et al., 2014). Thus, SBV was probably not present in northwestern Europe before 2011. The way it was introduced remains unknown. One hypothesis is transport by aircraft of infected midges, e.g. on flowers or on exotic animals from as yet unrecognized areas where SBV would be enzootic (Gale et al., 2015).

2.1.2 Structure

Viruses from the family Bunyaviridae are enveloped viruses with a segmented RNA genome.

SBV-virions are about 100 nm in diameter (Figure 1).

5 The genome of viruses in the Orthobunyavirus genus is made of three segments of negative- sense single-stranded RNA: L (large), M (medium) and S (small). These segments encode four structural proteins and two non-structural proteins:

- The S segment encodes the nucleoprotein N and the non-structural protein NSs;

- The M segment encodes a polyprotein, which is cleaved into two surface glycoproteins (Gn and Gc) and the non-structural protein NSm;

- The L segment encodes the RNA-dependent RNA polymerase (RdRp or L protein).

- In the virion, the genome is included in a ribonucleoprotein: each segment is associated to many copies of the nucleoprotein N and a few copies of the polymerase (Briese et al., 2013; Doceul et al., 2013; Wernike et al., 2014a).

Figure 1. Morphology of SBV: schematic drawing on the left, electronic microscopy on the right (bar = 100 nm).

RNA segments are associated with N nucleoproteins (not shown) and L proteins (in purple on the schematic drawing). Courtesy of Dr H. Granzow and M. Jörn, FLI.

The surface glycoproteins Gn and Gc are type I integral membrane proteins that are

embedded in the envelope. Their C-terminal parts, also known as cytoplasmic tails, are

towards the intraviral space while the N-terminal parts are in contact with the outer

environment. Their functions have not been fully elucidated but they are necessary for

budding of viruses of the genus Orthobunyavirus. They may be involved in virus fusion and

entry into the cells as well (Strandin et al., 2013).

6 The nucleoprotein N is the most abundant protein in the virion and in infected cells. Its major role is encapsidation of the genome but it may be involved in transcription and replication of the viral RNA (Wernike et al., 2014a).

In viruses from the genus Orthobunyavirus, the NSs protein is not essential for virus growth in vitro but is involved in viral pathogenesis. Functions of the NSm protein are still unknown, yet it may be participate in viral assembly (Eifan et al., 2013), probably in the Golgi apparatus (Doceul et al., 2013).

The L protein of bunyaviruses has a double activity: it acts both as an RNA polymerase and as an endonuclease. As an endonuclease, the L protein cleaves cellular messenger RNAs, leading to capped primers that initiate transcription of viral messenger RNAs; this process is known as ‘cap-snatching’ (Briese et al., 2013).

2.1.3 Phylogeny

The Orthobunyavirus genus belongs to the Bunyaviridae family. This family encompasses 350 viruses that are divided into 5 genera: Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. Viruses from the latter genus infect plants whereas viruses from the other genera infect vertebrates (Doceul et al., 2013).

The Orthobunyavirus genus is made of more than 170 viruses allocated to 18 serogroups (Doceul et al., 2013). The Orthobunyavirus genus includes viruses responsible for disease in humans, as Oropouche virus (Simbu serogroup), or Jamestown Canyon virus and La Crosse virus (California serogroup), and viruses responsible for disease in ruminants, as Aino virus and Akabane virus (Simbu serogroup) or Cache Valley virus (Bunyamwera serogroup) (Briese et al., 2013; Hoffmann et al., 2012a).

SBV was found to be closely related to viruses of the Simbu serogroup when it was

discovered. The most similar sequences were from Shamonda virus, Aino virus and Akabane

virus (AKAV), three viruses that can be found in cattle (Hoffmann et al., 2012b). None of

these viruses has been detected in continental Europe: Shamonda virus has been detected in

Nigeria, Japan and Korea; AKAV has been found in Australia, Japan, Korea, Israel, Saudi

Arabia, Kenya, Sudan, Cyprus and Turkey; Aino virus has been found in Japan, Korea and

Australia (Lievaart-Peterson et al., 2012).

7 Later, a phylogenetic study found the M segment was derived from Sathuperi virus (another virus of the Simbu serogroup) while the S and L segments were derived from Shamonda virus, suggesting SBV could be a reassortant between Sathuperi virus and Shamonda virus (Yanase et al., 2012). However, a subsequent study found SBV may be instead an ancestor of Shamonda virus, based on phylogenetic and serologic analyses (Goller et al., 2012). Part of the results of the latter phylogenetic analyses are reproduced in Figure 2.

Figure 2. Phylogenetic relationships of Simbu serogroup viruses for the M (A), L (B), and S (C) coding regions.

Maximum-likelihood trees (numbers at nodes: percentage of 1,000 bootstrap replicates, when >50). Scale bars:

nucleotide substitutions per site. GenBank accession numbers are shown in brackets. DOUV, Douglas virus;

SATV, Sathuperi virus; SBV, Schmallenberg virus; SHUV, Shuni virus; AINOV, Aino virus; SIMV, Simbu virus; PEAV,

Peaton virus; SANV, Sango virus; AKAV, Akabane virus; SABOV, Sabo virus; SHAV, Shamonda virus; OROV,

Oropouche virus. ND, not determined. Adapted from (Goller et al., 2012).

8

In addition to reassortment (genetic shift), bunyaviruses can evolve via point mutation

(genetic drift) (Briese et al., 2013). Genetic variability has been shown in SBV, with a

hypervariable region in the M segment corresponding to the glycoprotein Gc (Coupeau et al.,

2013; Fischer et al., 2013a; Hofmann et al., 2015; Rosseel et al., 2012). However, as yet, this

variability does not correlate with the time of collection, the geographical location, or the host

of the SBV strains (Fischer et al., 2013a; Hofmann et al., 2015).

9 2.2 Epidemiology

2.2.1 Susceptible species

EFSA has defined the species susceptible to SBV as the species in which SBV can replicate, with or without clinical signs (EFSA, 2014). ‘Receptive species’ means the same. According to EFSA, there are three categories of susceptible species to SBV:

- Species in which SBV has been detected in association with clinical signs;

- Species in which SBV has been detected (direct detection);

- Species in which antibodies against SBV have been detected (indirect detection) (EFSA, 2014).

2.2.1.1 Ruminants

In this manuscript, ‘ruminant’ is used in a broad sense, meaning any artiodactyl from the Ruminantia taxon or from the Tylopoda taxon (Camelidae).

2.2.1.1.1 Domestic ruminants

Herein are considered the usual domestic ruminants in Europe: cattle, sheep, and goats.

In all these species, direct and indirect detection of SBV and clinical signs in adults or in their offspring have been identified (van den Brom et al., 2012; Garigliany et al., 2012; Herder et al., 2012; Wernike et al., 2014a). The clinical signs will be detailed later in the manuscript (part 2.3).

2.2.1.1.2 Wild and exotic ruminants

SBV-infection has been detected in several species among wild and exotic ruminants.

Species in which SBV-RNA or antibodies have been detected are summarized in Table 1.

10

Table 1. Detection of SBV infection in wild and exotic ruminants from the wild or kept in captivity.

Species Antibodies against SBV

SBV

RNA Country Context Reference

Alpaca

(Vicugna pacos) x UK, Austria Captive animals (Jack et al., 2012)

(Steinrigl et al., 2014) Alpaca

(Vicugna pacos) x x Germany Experimental infection on

captive adult animals (Schulz et al., 2013) European bison

(Bison bonasus) x Poland Free-ranging animals (Larska et al., 2013a)

(Larska et al., 2014) Water buffalo

(Bubalus bubalis) x Turkey Captive animals (farm) (Azkur et al., 2013)

Red deer

(Cervus elaphus) x

Belgium, Italy, Poland , Austria,

UK

Free-ranging wild animals

(Linden et al., 2012) (Chiari et al., 2014) (Larska et al., 2013a) (Steinrigl et al., 2014) (Barlow et al., 2013)

(Larska et al., 2014) Sika deer

(Cervus Nippon) x Austria Not specified (Steinrigl et al., 2014)

Chamois (Rupicapra

rupicapra)

x Italy, Austria,

Spain Free-ranging wild animals

(Chiari et al., 2014) (Steinrigl et al., 2014) (Fernández-Aguilar et al.,

2014) Roe deer

(Capreolus capreolus)

x Austria, Belgium,

Spain, Poland Free-ranging wild animals

(Steinrigl et al., 2014) (Linden et al., 2012) (Fernández-Aguilar et al., 2014) (Larska et al., 2014) Fallow deer

(Dama dama) x Austria, UK,

Poland Free-ranging wild animals

(Steinrigl et al., 2014) (Barlow et al., 2013)

(Larska et al., 2014) Elk

(Alces alces) x Poland A free-ranging 6 month-old

calf (Larska et al., 2013a) Mouflon

(Ovis aries musimon)

x Poland Not specified (Larska et al., 2014)

Llama

(Lama glama) x x Germany Experimental infection on

captive adult animals (Schulz et al., 2013) Llama

(Lama glama) x Austria Not specified (Steinrigl et al., 2014)

11 In addition, in two zoological parks in the United Kindgom, antibodies against SBV have been detected in the following ruminant species: Bongo (Tragelaphus eurycerus), Banteng (Bos javanicus), Congo buffalo (Syncerus caffer), European bison (Bison bonasus), Gaur (Bos gaurus), Gemsbok (Oryx gazelle), Greater kudu (Tragelaphus strepsiceros), Moose (Alces americanus), Nile lechwe (Kobus megaceros), Pere David’s deer (Elaphurus davidianus), Reindeer (Rangifer tarandus), Roan antelope (Hippotragus equinus), Scimitar-horned oryx (Oryx dammah), Sitatunga (Tragelaphus spekii), and Yak (Bos mutus) (EFSA, 2014).

In spite of detection of SBV antibodies and RNA, clinical signs related to SBV infection have not been described in these wild and exotic species.

After experimental inoculation, alpacas and llamas showed a SBV RNAemia in the 9 days post infection (dpi) without clinical signs. SBV antibodies were first detected between 9 and 21 dpi (Schulz et al., 2013). In a field study in Germany, llamas and alpacas owners did not reported clinical signs or congenital malformations (Schulz et al., 2013).

A 6 month-old elk, found alone in a national park in Poland, showed weakness, hyperthermia hyperventilation, muscle tremors, and hind limb paresis: acute pneumonia was diagnosed. It died 5 days later despite treatment. Suppurative bronchopneumonia was found at necropsy and SBV RNA was detected in the serum. Bronchopneumonia and SBV infection were probably unrelated, yet the authors speculate SBV infection could have impaired the immune response in the elk, facilitating the pneumonia (Larska et al., 2013a). However, they did not report hematological or histological abnormalities to support the latter hypothesis.

2.2.1.2 Non-ruminants

As several viruses from the genus Orthobunyavirus can cause disease in humans, the possibility of SBV transmission from animals to humans was one of the most important question to answer at the beginning of the epizootics. Molecular and serological testing have been performed on exposed populations in Germany and in the Netherlands. SBV RNA and antibodies against SBV were not detected (Ducomble et al., 2012; Reusken et al., 2012). The

Reindeer (Rangifer tarandus)

x Austria Not specified (Steinrigl et al., 2014)

12 public health risk for SBV was concluded to be ‘absent or extremely low’ (Reusken et al., 2012).

In two zoological parks in the United Kingdom, antibodies against SBV have been detected in Onager (Equus hemionus) and Grevy’s zebra (Equus grevyi). However, SBV RNA has not been detected in samples from horse (Equus caballus) offspring tested in the Netherlands (EFSA, 2013).

Antibodies against SBV have been found in free-ranging wild boar (Sus scrofa) in Belgium (Desmecht et al., 2013). After SBV experimental infection, domestic pigs seroconverted but did not show clinical signs or SBV RNAemia (Poskin et al., 2014a), suggesting they are receptive to SBV though they do not develop disease.

SBV infection can occur in dogs, albeit few cases have been reported. In Sweden, antibodies against SBV have been found in one dog (Wensman et al., 2013). In France, SBV RNA was found in the brain of a puppy showing torticollis and degenerative encephalopathy while antibodies against SBV were detected in the mother (Sailleau et al., 2013a) (cf. appendix).

Nevertheless, in Belgium, a serologic survey did not detect any positive sample in 132 dogs (Garigliany et al., 2013). SBV infection is probably a rare event in dogs.

Finally, a mouse model of infection has been developed with the type I Interferon receptor knock-out (IFNAR

) mice. After experimental subcutaneous inoculation, SBV RNA was found in blood and organs; the mice lose weight and some died. Thus, this mouse strain is susceptible to SBV infection (Wernike et al., 2012a).

2.2.2 Transmission

2.2.2.1 Vectors

SBV is an arbovirus, which is “a virus which in nature can infect hematophagous arthropods by their ingestion of infected vertebrate blood. It multiplies in the arthropod’s tissues and is transmitted by bite to other susceptible vertebrates” (Mellor, 2000).

Biting midges, small blood-sucking insects of the genus Culicoides, are the putative vectors

for SBV. Their life cycle include eggs, larvae (four larval instars), pupa and adults (Carpenter

et al., 2013). The midges are usually active from April to October in the United Kingdom

13 (Tarlinton et al., 2012). The species in which SBV RNA has been found in Europe are C.

obsoletus complex (i.e. C. obsoletus sensu stricto and C. scoticus), C. dewulfi and C.

chiopterus (Elbers et al., 2013a, 2013b; Goffredo et al., 2013; Rasmussen et al., 2012, 2014;

De Regge et al., 2012). C. punctatus and C. nubeculosus may be involved as well (Balenghien et al., 2014; Larska et al., 2013b).

Vector competence has not been established under laboratory conditions for any of the aforementioned species, except for C. scoticus, because these species are hard to feed and to breed in the laboratory (Balenghien et al., 2014; Veronesi et al., 2013). Nevertheless, competence may be inferred from field data by two methods:

- By dissecting midges and performing SBV RT-qPCR on heads only: detection of SBV RNA in the head means the salivary glands are infected (De Regge et al., 2012);

- By analysis of the distribution of Cq-values after SBV RT-qPCR: a recent study showed a bimodal distribution of Cq-values indicated the ability for a species to be a vector (Veronesi et al., 2013).

Both methods have confirmed the vector competence of midges of the C. obsoletus complex, C. dewulfi and C. chiopterus (De Regge et al., 2012, 2014). These species belong to the subgenus Avaritia, known to breed on dung (Lühken et al., 2014) and bog land (Koenraadt et al., 2014).

A few studies have been carried out to elucidate the role of mosquitoes in SBV transmission.

No SBV RNA was found in 50,000 mosquitoes trapped in Germany in 2011 (Wernike et al., 2014b). Experimental oral infection of Aedes albopictus and Culex pipiens mosquitoes did not result in SBV replication to transmissible levels, suggesting these two species are unlikely to be true vectors of SBV (Balenghien et al., 2014).

The role of other arthropods as SBV vectors has not been explored.

2.2.2.2 Vertical transmission in ruminants

SBV can be transmitted vertically from the pregnant female to its offspring. Congenital

malformations associated to SBV RNA have been found in newborns, stillborn or aborted

animals in sheep, cattle, and goats (van den Brom et al., 2012; Garigliany et al., 2012; Herder

et al., 2012). Vertical transmission in other species has not been reported.

14 2.2.2.3 Horizontal transmission in ruminants

SBV RNA has been detected in fecal, oral and nasal swabs in subcutaneously-inoculated cows (Wernike et al., 2013a). Nevertheless, direct transmission of SBV from an infected ruminant to a naïve one by contact or by oral or nasal route is unlikely. Indeed, oral inoculation to cattle and nasal inoculation to sheep failed to produce RNAemia (Martinelle et al., 2015; Wernike et al., 2013a). Moreover, naïve cows in contact with viremic cows for 24 days did not show RNAemia and remained seronegative, as determined by SBV-specific ELISA (Wernike et al., 2012b).

Whether SBV can be sexually transmitted is still unknown. Bulls can excrete SBV in their semen, as shown by detection of infectious SBV in a few bovine semen samples from the field (Hoffmann et al., 2013; Ponsart et al., 2014; Schulz et al., 2014). However, the ability of the female to be infected by the vaginal route during artificial insemination or natural service has not been assessed. This topic deserves interest, as cows have been successfully infected with AKAV by uterine route at the time of artificial insemination (Parsonson et al., 1981a).

2.2.2.4 Overwintering

In May 2012 in France, eight months after the likely introduction date of SBV in the country (Zanella et al., 2013), evidence of acute infection was found in cows in France, suggesting SBV could overwinter or was reintroduced (Sailleau et al., 2013b). SBV overwintering was confirmed in Germany after acute infections that occurred in summer and fall 2012 (Conraths et al., 2013). SBV has then overwintered in 2013 and 2014, with new cases or SBV RNA detection being reported each vector season (Wernike et al., 2015).

Several mechanisms could account for SBV overwintering. First, SBV may persist in its host:

it seems unlikely at least in domestic ruminants, because viremia appeared to be short-lasting

in adults (Hoffmann et al., 2012b) and SBV RNA is not often detected in malformed

newborns (Bouwstra et al., 2013; De Regge et al., 2013). Characteristics of viremia in most of

the susceptible wild or exotic ruminants remain unknown. Second, transovarial transmission

of the virus in the vector might be involved in overwintering: one study reported SBV RNA in

nulliparous females of C. obsoletus complex and C. punctatus (Larska et al., 2013b). Third,

SBV may persist in adult midges during winter, as postulated for African Horse Sickness

15 Virus (Mellor, 2000). Adult midges of the C. obsoletus complex are able to survive, without blood meal, for 10 days at 4°C and up to 92 days at temperatures between 17°C and 25°C (Goffredo et al., 2004). As C. obsoletus midges have the ability to live indoors (endophagy) (Koenraadt et al., 2014), infected midges could thus survive in the coldest months in barns and infect vertebrates once the temperature rise. This hypothesis is supported by evidence of transmission of SBV to cattle in winter 2013 in Germany, after a rise in temperature above 5°C for a few days (Wernike et al., 2013b). Finally, as yet unidentified vectors could play a role in SBV transmission and overwintering.

2.2.3 Geographical repartition

From Germany, the Netherlands, and Belgium, where it was initially detected in 2011, SBV has then spread quickly to other European countries along the four cardinal directions (Figure 3) (Afonso et al., 2014), up to 320 km from the northern polar circle in Sweden (Chenais et al., 2013).

Figure 3. Regions (NUTS2) with at least one SBV herd confirmed by direct detection by period of first report (Afonso et al., 2014).

NUTS: Nomenclature of territorial units for statistics

16 In addition, antibodies against SBV have been found in 2013 in cows in Lithuania (Lazutka et al., 2014), and in cows and sheep in Greece (Chaintoutis et al., 2014), demonstrating presence of SBV in these countries.

Outside of Europe, in Turkey, SBV RNA was detected in aborted cattle and sheep fetuses in June 2012 (Yilmaz et al., 2014), suggesting a spread from northern Europe to Turkey. In this country antibodies to SBV have been found by ELISA in samples collected before 2011 (Azkur et al., 2013); however viral neutralization was not performed, thus cross-reactivity with other viruses from the Simbu serogroup cannot be excluded.

Finally, SBV might be circulating in Africa. Antibodies to SBV have been detected by ELISA in cattle, sheep and goats in Mozambique, in 2013; yet, the possibility of cross-reactivity with other viruses from the Simbu serogroup precludes a definitive conclusion (Blomström et al., 2014). Lesions retrospectively consistent with SBV infection have been found in sheep offspring in South Africa in 2006 and 2008, but tests specific for SBV detection were not available at this time (Leask et al., 2013).

2.2.4 Risk factors

The risk of SBV infection seems to differ among ruminant species. In wildlife, SBV seroprevalence was higher in heavy wild ruminants than in light wild ruminants in Poland in 2013 (Larska et al., 2014). Regarding domestic ruminants, within-herd prevalence was found to be lower in sheep than in cattle, in Germany (Helmer et al., 2015). In addition, goats have been shown to have a lower risk of SBV infection than sheep in Germany (Helmer et al., 2015).

These differences can be a consequence of intrinsic differences in susceptibility of these species to SBV. Experimentally, cattle needs a lower SBV infectious dose than sheep to be successfully infected (Poskin et al., 2014b; Wernike et al., 2012b).

Alternatively, the differences between species may depend on their difference of exposure to

midges. Culicoides spp. find their hosts after their odor (Koenraadt et al., 2014) and have

developed host preferences. Cattle was found to be more attractive than sheep for midges of

the C. obsoletus complex in Germany (Ayllón et al., 2014). Housing conditions may also

facilitate exposure of some species to midges. Indeed, the moist cesspool under slatted floors

17 in cow barns may offer breeding sites for Culicoides spp. while sheep barns display most often deep litter, which may not favor midges larval development (Helmer et al., 2015).

For a given species, breeding conditions are also a risk factor for SBV infection. Indoor dairy cow herds showed lower SBV seroprevalence of SBV than outdoor herds, probably due lower midge exposure of cattle kept indoors (Tarlinton and Daly, 2013; Veldhuis et al., 2014a). The housing conditions were also found to influence seroprevalence in goats, with the outdoor herds tending to have a higher within-herd seroprevalence in France and in Germany (Helmer et al., 2013; Valas et al., 2014).

Other factors have been found to influence the probability of domestic ruminants from a farm to be infected by SBV. According to a study in the Netherlands, presence of one or more dogs in the farm, on the one hand, and purchase of silage, on the other hand, were found to be associated with an increased odd of malformations in newborn lambs (Luttikholt et al., 2014).

As shown previously, dogs may be infected by SBV but viremia in this species has not been

characterized, thus their role in SBV dissemination is still unknown. The biological link

between the purchase of silage and SBV-associated malformations remains unclear.

18 2.3 Clinical signs and lesions in affected animals

In this section, the clinical signs and lesions associated with SBV infection in domestic ruminants will be described and compared with those involving two other members of the genus Orthobunyavirus that infect domestic ruminants: AKAV and Cache Valley virus (CVV).

2.3.1 Non-pregnant adults

2.3.1.1 Clinical signs

At first, SBV infection was discovered in non-pregnant dairy cows in Germany. It was responsible for short-lasting non-specific clinical signs, namely fever, decreased milk production, and diarrhea (Hoffmann et al., 2012b). One report suggests that goats infected by SBV may also experience diarrhea and reduction in milk yield (Helmer et al., 2013). Fever, diarrhea and decreased milk production have been sometimes reported in sheep from the field, without an obvious causal link with SBV infection (Lievaart-Peterson et al., 2012; Luttikholt et al., 2014).

Likewise, AKAV infection can result in subclinical disease in adult ruminants (Spickler, 2009) and in a drop of milk production in dairy cows (Horikita et al., 2005). However, more severe clinical signs have been reported in adult cattle. In 2000 and 2010 in Korea, outbreaks of neurological signs associated with AKAV infection occurred in 2 to 7 years-old and in 4 to 72 months-old cows, respectively. The clinical signs included hypersensitivity, tremors, locomotor ataxia, and lameness (Lee et al., 2002; Oem et al., 2012). No clinical signs have been reported in association with CVV infection in adult ruminants.

2.3.1.2 Lesions

No lesions have been described in association with SBV infection in non-pregnant adult

ruminants in the field.

19 During the outbreak of neurological signs associated with AKAV infection in Korea in 2000 in adult cows, 5 affected cows were slaughtered and submitted to post-mortem examination.

They were devoid of gross lesions, but histological examination revealed similar lesions in their brains and spinal cords. The brain, especially the pons and medulla oblongata, was characterized by moderate to severe perivascular infiltrations of lymphocytes, macrophages, and plasma cells with multifocal gliosis and neuronal necrosis. In the spinal cord, similar lesions were found in the ventral horns with lymphohistiocytic perivascular cuffing, gliosis, and neuronal necrosis and loss. Overall, these lesions corresponded to a non-suppurative encephalomyelitis (Lee et al., 2002).

Phylogenetic analyses showed the AKAV strain responsible for this Korean outbreak was closely related to Iriki strain, an AKAV strain that has been associated earlier with encephalitis in adult cattle in Japan (Oem et al., 2012). Despite the absence of lesions in adults associated with SBV infection, up to now, we cannot exclude that new strains with the ability to trigger encephalitis in adults may emerge in the future.

2.3.2 Pregnant females and their offspring

After discovery of SBV in non-pregnant adult cows in Germany, epizootics of congenital malformations were reported in sheep in the Netherlands. They were associated in about half of the cases with detection of SBV RNA in the brains from the affected newborns (van den Brom et al., 2012), showing the ability of SBV to cross the placenta in pregnant ewes and to cause lesions in the growing embryo or fetus.

2.3.2.1 Clinical signs in pregnant females and in newborns

At parturition, pregnant females infected by SBV can show clinical signs of dystocia when

their offspring is malformed (van den Brom et al., 2012). During pregnancy, there are several

lines of evidence SBV could be associated with embryonic or fetal death and abortion. In

flocks affected by SBV, increased numbers of repeat breeders and increased rate of abortions

suggest early embryonic or fetal loss and abortions in ewes (Dominguez et al., 2014; Lievaart-

20 Peterson et al., 2012; Luttikholt et al., 2014; Saegerman et al., 2014) and goats (Dominguez et al., 2014; Helmer et al., 2013). In cattle flocks affected by SBV, increased numbers of repeat breeders and of early embryonic deaths have been reported as well (Dominguez et al., 2014).

All the aforementioned data come from retrospective studies. In addition, one report describes an association between early fetal death (around 60 days of gestation (dg)) in two cows and SBV genome in the corresponding allantoic fluids (Steinrigl et al., 2014).

The birth of both one malformed and one healthy offspring has been described in SBV- infected cattle (Wernike et al., 2014c) and sheep (van den Brom et al., 2012). In the affected sheep flocks, the lambs were either malformed, dummy with inability to suckle or normal (van den Brom et al., 2012). Neurological signs have also been described in a newborn calf infected in utero by SBV: hypertonicity, hyperreflexia, depression, blindness, ventrolateral strabismus, and inability to stand (Garigliany et al., 2012). These neurological signs are suggestive of SBV-induced lesions in the central nervous system (CNS).

During an outbreak of AKAV infection of pregnant cows in Japan in 1973-1974, a succession of different clinical signs were noticed. Abortions, stillbirths, premature births and neonatal deaths were seen at the beginning of the outbreak. Then, the newborn calves were alive but displayed musculoskeletal malformations as torticollis and arthrogryposis. At the end of the outbreak, the newborn calves showed blindness or tongue paralysis (Konno et al., 1982).

Similarly, infection of sheep with CVV in the field was associated with stillbirths, mummified fetuses, malformed fetuses or newborns, and weak lambs as well (Edwards et al., 1989).

2.3.2.2 Lesions in fetuses and newborns

A wide range of lesions have been described in fetuses and newborns with proven or presumptive infection by SBV during gestation (Table 2). The most common lesions affect the skeletal muscle, the CNS and the axial skeleton; they can occur together in combination (Herder et al., 2012; Peperkamp et al., 2014; Seehusen et al., 2014). The animals may show arthrogryposis (Figure 4) associated with histological evidence of muscular hypoplasia (Figure 4), characterized by a reduction in number and diameter of the myofibrils, with or without loss of cross-striation in myofibrils and fatty replacement (Herder et al., 2012;

Seehusen et al., 2014). In the CNS, the most common lesions are hydranencephaly,

porencephaly, hydrocephalus, cerebellar hypoplasia and micromyelia (Figure 4); they are

21 sometimes associated with nonsuppurative inflammation and neuronal degeneration and necrosis (Figure 4). The animals often display vertebral malformations, including lordosis, kyphosis, scoliosis, and torticollis, and may show brachygnathism inferior (Figure 4) (Herder et al., 2012).

Table 2. Lesions in fetuses and newborns associated with spontaneous SBV infection in domestic ruminants.

Gross lesions Histological lesions Ref.

Bovine Porencephaly, hydrocephalus, hydranencephaly, brain stem hypoplasia, cerebellar hypoplasia, cerebellar dysplasia, micromyelia, arthrogryposis,

torticollis, lordosis, scoliosis, kyphosis, cranial malformations, brachygnathism inferior, prognathism, ectopia cordis, lung hypoplasia,

ventricular septal defect

Non-suppurative meningoencephalitis, non- suppurative poliomyelitis, skeletal muscle hypoplasia, lymphoid depletion in thymus

and lymph node, chronic hepatitis

(Garigliany et al., 2012) (Peperkamp et al., 2012) (Herder et

al., 2012) (Seehusen et al., 2014) (Peperkamp et al., 2014) (Bayrou et

al., 2014) Ovine Arthrogryposis, torticollis, lordosis, scoliosis,

kyphosis, brachygnathism inferior, domed skull, flattened skull, hydranencephaly, hydrocephalus, micrencephaly, macrocephaly, brainstem hypoplasia, cerebral hypoplasia, cerebellar hypoplasia, cerebellar dysplasia, micromyelia, cardiac ventricular septal

defect, unilateral hydronephrosis, colonic atresia

Non-suppurative meningoencephalitis, skeletal muscle hypoplasia, lymphoid depletion in spleen or lymph

node, cataract, decreased hematopoietic cellularity in

bone marrow

(van den Brom et al.,

2012) (Herder et

al., 2012) (Peperkamp et al., 2014) Caprine Arthrogryposis, vertebral deformities,

brachygnathism inferior, hydrocephalus, porencephaly, cerebellar hypoplasia, lung hypoplasia

Non-suppurative meningoencephalitis, non-

suppurative poliomyelitis

(Herder et al., 2012) (Wagner et

al., 2014)

22

Figure 4. Gross and histological lesions from SBV-infected fetuses (Herder et al., 2012).

(1) Aborted ovine fetus with arthrogryposis, torticollis, and brachygnathism inferior. (2) Cerebellar hypoplasia in a bovine fetus. (3) Brain from a bovine fetus. Hydrocephalus and porencephaly (arrow) in the adjacent cerebral parenchyma. (4) Brain; goat. Perivascular lymphocytes and macrophages. Hematoxylin-Eosin (HE). (5) Brain;

goat. Glial nodule of microglia/macrophages. HE. (6) Brain; sheep. Chromatolysis, with dispersion of Nissl substance, swelling, and homogeneous cytoplasmic eosinophilia of a neuron. HE. (7) Brain; calf.

Hypereosinophilic, necrotic neuron. HE. (8) Cervical spinal cord; calf. Micromyelia with severe reduction of gray matter and few neurons in one ventral horn (arrow). Ventral meningeal fibrosis (asterisk). HE. (9) Skeletal muscle; sheep. Myofibrillar hypoplasia with few residual normal muscle fibers (asterisk). HE.

The nature of the musculoskeletal and nervous lesions associated with SBV are similar in

cattle and sheep. Still, the severity of the CNS lesions may be higher in lambs than in calves

23 (Peperkamp et al., 2014). The lesions have not been as extensively described in goats as in cattle and sheep.

Gross and histological lesions associated with AKAV infection in domestic ruminants are very similar to those described above for SBV. They are detailed in Table 3. The CNS, the skeletal muscle and the axial skeleton are again the most affected systems.

Table 3. Lesions in fetuses and newborns associated with AKAV infection in domestic ruminants.

Context Gross lesions Histological lesions Ref.

Bovine Spontaneous infection

Arthrogryposis, scoliosis, kyphosis, lordosis, torticollis,

porencephaly, hydranencephaly

Muscle hypoplasia or atrophy, decrease in the number of neurons in

the ventral horns in the spinal cord, nonsuppurative encephalomyelitis,

retinal atrophy

(Konno et al., 1982) (Ushigusa et al., 2000)

Experimental infection

Same as above (normal fetuses were also found)

Muscle degeneration and atrophy, decrease in the number of neurons in

the ventral horns in the spinal cord, suppurative meningoencephalitis,

cerebellar dysplasia

(Kirkland et al., 1988)

Ovine Experimental infection

Arthrogryposis, scoliosis, kyphosis, lordosis, torticollis,

porencephaly, hydranencephaly, dwarfism,

brachygnathism inferior, micrencephaly, lung hypoplasia (normal fetuses

were also found)

Muscle atrophy and degeneration, myelitis and/or neuronal loss in the ventral horns in the spinal cord, meningoencephalitis and/or cysts

and malacia in the brain, subependymal rosettes in the brain,

thymic depletion

(Hashiguchi et al., 1979) (Parsonson et al., 1977)

(Parsonson et al., 1981b) (Narita et al., 1979) Caprine Experimental

infection

Arthrogryposis, scoliosis, torticollis, hydranencephaly

Degeneration and necrosis in skeletal muscle and/or myositis, non-

suppurative encephalitis

(Kurogi et al., 1977) (Konno and

Nakagawa, 1982)

Likewise, the lesions described in the offspring of SBV-infected sheep in the field include

lesions of the skeletal muscle (arthrogryposis with skeletal muscle hypoplasia), of the CNS

(hydrocephalus, hydranencephaly, micrencephaly, cerebellar hypoplasia, micromyelia with

loss of motor neurons, porencephaly), and spinal column deformities (Edwards et al., 1989).

24 In addition, oligohydramnios, i.e. the close apposition of the amniotic membrane to the fetal body, was described after experimental in utero inoculation of CVV to pregnant ewes at 35 dg (Hoffmann et al., 2012a). The latter lesion may however rely upon the route of inoculation.

2.3.3 Hypotheses on the pathogenesis of the lesions in fetuses and newborns

Previous works on CVV and AKAV have brought pieces of information about the pathogenesis of the viral-induced lesions. Given that SBV is phylogenetically close to these viruses and causes very similar clinical signs and lesions, it is likely they are involved in common mechanisms of disease. By analogy with these virus, it is thus possible to formulate hypotheses regarding the pathogenesis of the lesions induced by SBV.

2.3.3.1 Factors influencing the development of lesions in fetuses and newborns

From the literature, both the age of the conceptus (i.e., the embryo or fetus with its placenta) and the inoculum have an impact on the development of lesions in fetuses and newborns.

2.3.3.1.1 Influence of the age of the conceptus at the time of infection

In cattle, an experimental infection with AKAV showed a sequential development of lesions depending on the stage of pregnancy at which the cow was infected. Hydranencephaly and porencephaly developed in fetuses after infection between 76 and 104 dg. Arthrogryposis developed in fetuses infected later, when infection took place between 103 and 174 dg. No lesions were found in fetuses born from cows infected before 76 dg (Kirkland et al., 1988). As the authors suggested, the absence of fetal lesions before 76 dg may be explained by immaturity of the placentomes until this day, with subsequent isolation and protection of the conceptus from the virus (Kirkland et al., 1988). However, they could not monitor the early embryonic development, thus they could not exclude embryonic mortality when infection took place before 76 dg (Kirkland et al., 1988).

Similarly, in sheep, the lesions that developed seem to depend on the age of the conceptus at

the time of infection with AKAV. A study from Japan showed that, when experimental

25 infection occurred between 29 and 45 dg, newborns displayed arthrogryposis and hydranencephaly. After inoculation between 30 and 70 dg, abnormal newborns were either weak, with or without porencephaly, or dwarf; there were also stillborn lambs. However, if the inoculation took place between 90 and 100 dg, all the newborns were normal (Hashiguchi et al., 1979). Another experimental infection of pregnant ewes with AKAV showed that brain lesions (necrosis and gliosis) could still occur after inoculation at 90 dg (Narita et al., 1979).

One study showed that the ovine fetuses infected transplacentally could produce neutralizing antibodies against AKAV as soon as 64 dg (Hashiguchi et al., 1979). The development of the immune system in the fetus may inhibit viral progression and subsequent lesions; therefore it may be one reason for the lack of lesions at late stages of gestation. However, the immune system activity may not be the only factor responsible for this lack of lesions. An experimental inoculation of AKAV intraperitoneally in ovine fetuses at 120 dg resulted in a strong immune response, yet the fetuses showed damage in brain and skeletal muscle (McClure et al., 1988). The authors suggested that the stage of maturity of the target-organs may be of greater importance in determining its susceptibility to virus-induced damage than the fetal immune response (McClure et al., 1988).

The susceptibility window of goat fetuses to AKAV infection has been partially described. An experimental infection of a pregnant goat by AKAV at 40 dg resulted in one malformed fetus but a normal twin fetus. The malformed fetus displayed hydranencephaly, arthrogryposis, scoliosis, and torticollis (Kurogi et al., 1977). Another experimental infection at 30 and 60 dg led to degeneration and necrosis in the skeletal muscle and/or myositis in all fetuses at 10 dpi.

Only the fetuses infected at 60 dg showed also non-suppurative encephalitis (Konno and Nakagawa, 1982).

In summary, the susceptibility of the growing embryo or fetus to AKAV infection may depend on:

- The maturity of the placentomes: if the infection of the pregnant female takes place before maturity; the conceptus may be protected from viral invasion;

- The stage of maturity of the target-organs: with increasing maturity, the target organs

may be no more susceptible to the virus;

26 - The stage of development of the fetal immune system: with increasing development, the immune system could inhibit the progression of the virus and the virus-induced damage.

By analogy with AKAV, hypotheses can be drawn on the effects of SBV infection in pregnant females depending on the stage of gestation. Figure 5 shows the putative consequences of SBV infection in pregnant ewes and goats.

Figure 5. Hypothetic consequences of SBV infection in pregnant goats and ewes depending on the stage of gestation.

By analogy with data from experimental infection with AKAV (Hashiguchi et al., 1979; Konno and Nakagawa, 1982; Narita et al., 1979). AI: artificial insemination; CNS: central nervous system; dg: day of gestation; SKM:

skeletal muscle.

2.3.3.1.2 Influence of the inoculum

Several teams have performed AKAV inoculations in pregnant ewes and their results

sometimes differed. As an example, one study showed that lesions in fetuses occurred only if

the inoculation had taken place between 30 and 36 dg but not between 38 and 82 dg

(Parsonson et al., 1977), while another study showed fetal lesions after inoculation between

29 and 70 dg (Hashiguchi et al., 1979). In these cases, the protocols were similar regarding

the route of inoculation (intravenous), but they differed in the strains used as well as in the

number of passages and the host species used for passage. The authors of the second study

hypothesize that the difference in results could rely upon the difference in virulence between

the two inocula, depending on the viral strain and the history of passages (Hashiguchi et al.,

27 1979). Moreover, the viral load of the inoculum could also exert an influence on the severity of the damage in fetal tissues.

2.3.3.2 Mechanisms involved in the development of lesions at the tissue level

2.3.3.2.1 Microscopic lesions and correlation with tissular and cellular tropism

In one study, pregnant ewes were inoculated at 35 dg with CVV and were sequentially slaughtered between 42 and 63 dg. The results suggest two hypotheses about the mechanism of lesion formation.

First, this study shows a progression of the microscopic lesions in the fetuses along with the course of infection between 7 and 28 dpi. Between 7 and 10 dpi, the brain displayed necrosis in matrix and intermediate zones of the cerebral cortex and brainstem; necrosis was also noticed in the dorsal horns of the spinal cord and in the skeletal muscle, without vascular lesions. At 14 dpi, hydrocephalus ex vacuo was noticed, and myositis with mononuclear cells and granulocytes was seen in skeletal muscle. Then, between 21 dpi and 28 dpi, the histological lesions were hydrocephalus ex vacuo, myositis with muscle hypoplasia, and micromyelia (Hoffmann et al., 2012a). This sequential progression suggests that hydrocephalus ex vacuo, and probably hydranencephaly, proceed from necrosis of progenitor cells in the brain.

Second, the microscopic lesions were associated with the presence of CVV in the affected tissues, i.e. the skeletal muscle and the CNS. Viral RNA and antigen, as determined by ISH and IHC, were associated with necrotic foci in muscle, brain and spinal cord. The target cells were progenitor cells in the periventricular area in the brain, and myofibers in the muscle;

positive cells were also found in the spinal cord but their nature was not identified (Hoffmann et al., 2012a). This association between lesions, viral RNA and viral antigen are suggestive of a direct effect of the virus in the aforementioned tissues.

The pathogenesis of skeletal muscle hypoplasia (and the associated arthrogryposis) is a matter

of debate. In calves with arthrogryposis, a relationship has been described between the lesions

observed in spinal cord and the side(s) affected by arthrogryposis: bilateral depletion of the

ventral horn neurons was associated to bilateral arthrogryposis while unilateral depletion of